JP2882775B2 - Human-glia-derived neurite factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] TECHNICAL FIELD [0001] The present invention relates to a glial cell.
Neurite-promoting factor (neurite-pr
Omoting factor), neurite-promoting activity
Maintaining related proteins and their fragments, neurites
DNA encoding amino acid sequence of promoter and fragment thereof
Fragment, a hybrid vector containing such DNA,
Inns transformed with hybrid vectors such as
Mainly, production of the DNA vector and transformed host
Methods, neurite-promoting factors, related proteins and fragments thereof
For producing nervous system injury
About their use. [0002] 2. Description of the Related Art Glial cells cause damage to the nervous system.
And perform essential functions in the subsequent course
Can be About the nature of such glial-nerve interactions
Knowledge of macromolecules present in trace amounts in vivo
Need to be determined. Cultured rat glioma cells
Giant components promote neurite outgrowth in neuroblastoma cells
Release the child. Glial-derived neurite promoting factor in this rat
Child (glia-derived neurite-promoting factor; GdNPF)
Have been identified and characterized [J. Guenther,
 H. Nick and D. Monard,EMBO J.,4, 19
63-1966 (1985)]. [0003] This is due to neurite outgrowth, as well as urokina.
Ase, tissue plasminogen activator, thrombi
Of serine proteases such as glycine and trypsin
The apparent molecular weight of 43,000 which causes both
Protein. Protease and rat GdNPF
Formation of sodium dodecyl sulfate resistant complex between
Indicated by the same author. Rat GdNPF free
Or plasminogen activator associated with tumor cells
Inhibits the activity of
Vesicle neuron (granule cell neuro)
n) prevent movement. [0004] Induce neurite outgrowth and serine pro
Such neurite-promoting factors that inhibit theases
Promotes nerve fiber regeneration after transsystem injury and is normal
It is expected to interfere with cell and tumor cell migration.
However, human therapy of known rat GdNPF
Application is expected for rat GdNPF in humans
Relentlessly disturbed by antigenicity. The problem is human Gd
This can be overcome by using NPF. [0005] SUMMARY OF THE INVENTION One object of the present invention
Is to provide human GdNPF. GdNPF,
Fragments that maintain neurite-promoting activity, and GdN
The problem of industrial synthesis of PF-related peptides is recombinant DNA technology
Is solved by Another object of the present invention is to provide a GdNPF,
Methods for producing GdNPF-related peptides and fragments thereof;
GdNPF, GdNPF-related peptide or fragment thereof
To provide a medicine containing [0006] SUMMARY OF THE INVENTION The present invention is particularly directed to pure
Toglia-derived neurite-promoting factor (GdNPF), and
And related peptides that maintain neurite-promoting activity
And fragments. These compounds are responsible for neurite outgrowth and
Causes both serine protease inhibition. Further details
Alternatively, the present invention provides the following formula (I): [0007] Embedded image[In the formula, Cys may be disulfur in some cases.
Exists in the form of an arrow; X₁Is hydrogen, an acyl group such as phor
A mill group or an alkanoyl group such as a palmitoyl group,
Ristoyl or lower alkanoyl groups such as acetyl
Or a propynyl group, or the following formula (II): Met (-1
9) -Asn-Trp-His-Leu (-15) -Pro-Leu-Phe-Leu-Leu (-10) -A
la-Ser-Val-Thr-Leu (-5) -Pro-Ser-Ile-Cys (-1)-
Peptide residues, or 1-1 from the carboxy terminus
A fragment of the residue of formula (II) comprising 8 amino acids
Therefore, these peptide residues may be acylated
And X_TwoIs Arg or Thr-Gly
Which are glycosylated,
Human GdNPF; one or more of the compounds of formula (I)
A plurality, in particular one, two, three or four single aminos
Acid is replaced by other amino acids to promote neurites
Related polypeptides that retain their sex;
Amino acid chain between amino acid and amino acid at position 378
10 or more consecutive amino acids selected from
Depending on one or more, for example one, two or three
Fragment of a compound of formula (I) comprising two other amino acids
About. [0009] BEST MODE FOR CARRYING OUT THE INVENTION GdNPF of the formula (1)
And may have no carbohydrate residues.
You may not. Typically, the glycosylated formula (I)
GdNPF has one or more carbohydrate residues, for example,
N-glycosidic bond to asparagine (Asn) residue
N-acetylglucosamine or N-acetylglucosami
-Containing oligosaccharides and / or serine (Se
r) residue or threonine (Thr) residue with O-glycosylation
N-acetylgalactosamine or NA
Contains oligosaccharides containing cetyl galactosamine
You. In the GdNPF of the formula (I),
Residues may be present in reduced form as shown
In good or oxidized form, i.e. the disulfide form
Thus, an SS bridge may be formed.
Is the intramolecular S between any two Cys residues of formula (I)
-S bridge is formed. Acyl group X₁Is in the natural protein
It can be any acyl group found. Especially,
Acyl group X₁Is a lower alkanoyl group such as an acetyl group
Or a formyl group, preferably an acetyl group. The peptide residue X of the formula (II)₁Is a signal
It is a pseudo. GdNPF when formed in ribosome
Is the total peptide residue X of formula (II)₁It contains. After translation
Processing cleaves this signal peptide or part thereof.
Separate. In the present invention, X is a peptide residue of the formula (II)
Or a fragment of this residue as defined above, especially the amino acid
A fragment consisting of 18--1, Pro-Ser-Ile-Cys-(-4 to-
1), Ser-Ile-Cys-(-3 to -1), Ile-Cys-, or Cy
For a GdNPF of formula (I) that is only s-. Formula (II)
Peptide residues, or 1-18 from the carboxy terminus
Of amino acids at its N-terminus.
Silylated, e.g., acetylated or formylated
Good. R. W. Scott et al. [J. Biol. Chem.,26
0, 7029-7034 (1985) discloses a protease
Describes a polypeptide called nexin
doing. This serine protease inhibitor was cultured
Released from human fibroblasts. Protease Nexin
Has an apparent molecular weight of 43,000 and has the formula (I)
2 identical to the N-terminal 28 amino acids of GdNPF
Has an N-terminal amino acid sequence consisting of 8 amino acids
It has been reported. However, protease
Published protein called kucin and GdNP of the present invention
There is clear evidence that F is a different compound
You. This known protein, which is expected from
Amino acid composition, and X₁GdNP of the formula (I) wherein
Table 1 shows the amino acid composition of F. Threonine (Th
r), serine (Ser), glutamic acid and glutamine
(Glu / Gln), proline (Pro), valine (V
al) and isoleucine (Ile)
Also noticeable differences are found. [0013] [Table 1] The present invention further maintains neurite promoting activity.
A GdNPF-related polypeptide, eg, one or
Multiple single amino acids replaced by other amino acids
Including the compounds of formula (I) Such related ports
Ripeptides are naturally mutated or altered at the DNA level
By chemically induced mutation or by chemical synthesis
It can be formed by amino acid substitution. this
Such related polypeptides further include human GdNPF
Hybrid poly consisting of fusion fragments from different animal species
And peptides. The fragment of the present invention may be, for example,
Of a compound of formula (I) in which two amino acids have been deleted
Fragments, for example, amino acids 2 to 378, 3 to
The amino acid at position 378, the amino acid at positions 4 to 378,
5th to 378th amino acids, 6th to 378th amino acids
Including amino acids, or amino acids at positions 7-378
Fragments or amino acids³¹⁰Arg and amino acids
³¹¹Ser and / or³⁴⁵Arg and³⁴⁶Ser and
Optionally from 10 to 50 comprising other amino acids
Amino acids (an essential part of the serine protease substrate)
Is a small fragment consisting of
You. Another preferred fragment is antithrombin-
Shows considerable homology with III or α1-antitrypsin
Fragments containing the region, for example, positions 72-96, 13
4th to 146th or 159th to 195th, and
Amino acids selected from the amino acid chains at positions 314 to 378
Acid, and optionally one or more, such as 1
And fragments comprising two, three or two other amino acids.
No. Between the amino acids 321 and 334
A fragment consisting of 14 amino acids, ie, the formula: Gln-Lys-
Ala-Lys-Ile-Glu-Val-Ser-Glu-ASp-Gly-Thr-Lys-Ala
An additional fragment; between amino acids 322 and 334
Consists of 13 amino acids and an N-terminal histidine residue
Fragment, that is, the formula: His-Lys-Ala-Lys-Ile-Glu-Val-Se
a fragment of r-Glu-Asp-Gly-Thr-Lys-Ala;
Add 10 amino acids and carboxy terminus between positions 54
A fragment consisting of additional -Ser-Phe residues, ie the formula: Arg-Ser-
Fragment of Ser-Pro-Pro-Trp-Phe-Ile-Val-Asp-Ser-Phe;
And 14 amino acids between amino acids 175 and 188
A fragment consisting of amino acids, ie the formula: Lys-Ser-Arg-Phe-
Gln-Pro-Glu-Asn-Thr-Lys-Lys-Arg-Thr-Phe
Preferred. Human GdNPF, Related Peptides and Their
Fragments from cells that produce the desired compound.
Or by synthesis by condensation reaction
it can. For example, human GdNPF is the nerve that produces it.
Glioma cells or other glial systems
Cells are transferred to a suitable medium, such as minimal essential medium, Dulbecco's.
In a modified Eagle medium, PRMI 1640 medium, etc.,
Optionally, whole serum, such as fetal calf serum and / or
Growth-promoting compounds, mitogens, antibiotics and other supplements
It is obtained by supplementing the substance and culturing. Desired
GdNPF is isolated and studied in a conventional manner, for example, below.
Purify by the method. Human GdNPF, related peptides, and especially
The fragment can also be obtained by chemical methods, for example, M. Bodansz
ky,Principles of Pepride Synthesis, Springer-Ver
synthesized by the condensation reaction described in Lag, 1984.
It is possible. Fragments are synthesized, for example, by solid phase methods.
In this method, the amino group is protected amino group
The acid is bound to a suitable resin, the protecting groups are removed,
The protected second amino acid is replaced with the amino group of the first amino acid.
And the next step after deprotection and amino group protection.
Cycles of condensation with amino acids are carried out with peptide residues of desired composition.
Is completed, and finally this peptide residue
Is cleaved from the resin and deprotected. In particular, human GdNPF, related peptides and
The fragment can be used, for example, to transform the transformed host
Culture under conditions that permit expression of the peptide, and
A recombinant DNA technique comprising isolating a compound of formula
Can be prepared. More specifically,
The compound is (A) Glial cell cDNA library or genome
DNA encoding GdNPF from DNA library
Or isolating a fragment thereof and optionally isolating the DNA.
Mutating or chemically synthesizing such DNA
And; (B) introducing the DNA into an appropriate expression vector; (C) transforming the resulting hybrid vector into a recipient host
Convert; (D) Culture under conditions where only the transformed host survives
Transformed from an untransformed host
Select; (E) transforming the transformed host with a heterologous polypeptide;
Culturing under conditions permitting expression; and (F) human GdNPF or its related peptide or
Isolating the fragment. [0021] Recombinant DNA techniques for these peptides
The steps involved in manufacturing will be discussed in more detail later. Human
For the production of GdNPF, live cDNA of step (a)
The rally is preferably from human glioma cells, eg, "Cl
lection Nationale de Cultures de Microorganismes "
 , Pasteur Institute in Paris on 5 February 1986
o. Human glioma (gli) deposited as I-518
oma) Derived from the cell line. Genome D of step (a)
NA libraries were prepared from human placenta or human fetal hepatocytes.
Can be manufactured. Related peptides such as rat Gd
For the production of NPF, other glial cells, such as
Gut cells, especially C6 rat glioma cells
Thus, a cDNA library can be prepared. [0022] The present invention also provides glial-derived neurite promotion.
DNA encoding factor (GdNPF), for example, human G
DNA encoding dNPF or rat GdNPF,
One or more of these variants, e.g., one,
DN having 2, 3, or 4 nucleotide mutations
A, Related polypeptide maintaining neurite-promoting activity
DNA encoding 15 or more nucleotides
And fragments of these DNAs comprising these
DNA is understood to be single-stranded or double-stranded. In particular,
The invention of the following formula (III): [0023] Embedded image[0024] Embedded image[Wherein Y₁Is alanine (Ala)
And is GCT, GCC, GCA or GCG; Y_TwoIs al
Ginin (Arg), and encodes CGT, CGC, CGA, CGG,
AGA or AGG; Y_ThreeIs asparagine (Asn)
Encoding and is AAT or AAC; Y_FourIs ass
Encodes formic acid (Asp) and GAT or G
AC; Y_FiveEncodes cysteine (Cys),
And TGT or TGC; Y₆Is glutamine (G
ln) and is CAA or CAG; Y
₇Encodes glutamic acid (Glu) and GAA
Or GAG; Y₈Codes for glycine (Gly)
And GGT, GGC, GGA or GGG; Y₉Is histi
Encodes gin (His) and in CAT or CAC
Yes; Y_TenCodes for isoleucine (Ile)
And ATT, ATC or ATA; Y₁₁Is leucine
(Leu) and TTA, TTG, CTT, CTC, CTA or
Is CTG; Y₁₂Encodes lysine (Lys), and
Y is AAA or AAG;₁₃Is methionine (Me
t) and is ATG; Y₁₄Is phenyl
Encodes alanine (Phe) and TTT or TT
C; Y₁₅Encodes proline (Pro) and
Y is CCT, CCC, CCA or CCG;₁₆Is serine (Se
r) and encode TCT, TCC, TCA, TCG, AGT or AGC
And Y₁₇Encodes threonine (Thr) and
ACT, ACC, ACA or ACG. Y₁₈Codes for tryptophan (Trp)
And TGG; Y₁₉Is tyrosine (Tyr)
Encoding and is ATA or TAC; Y₂₀Is Bali
(Val) and GTT, GTC, GTA or GTG
And Y_{twenty one}Is the stop codon TAA, TAG or TGA; Y
_{twenty two}Is Y_TwoOr Y₁₇-Y₈And Z₁Is a promoter
12 or more nucleotides containing the sequence
Flanking DNA residues; Z_TwoDoes not exist
How or one or more nucleotides
Flanking DNA sequences comprising:₁And Z
_TwoAre connected in some cases.) Encodes the human GdNPF represented by
DNA, DNA of formula (III) and its complementary DN
A) (where adenine (A) is
Binds with Min (T) and vice versa, and guanine
(G) binds cytosine (C) and vice versa
), The complementary DNA itself, one or more
These variants in which the nucleotides are mutated, as well as 1
Of these DNAs comprising 5 or more nucleotides
Regarding fragments. The invention particularly relates to the following formula (IV): [0029] Embedded image [0030] Embedded image[0031] Embedded image(Where W is A or ACAG, X_Two
Is Arg or Thr-Gly; and Z_ThreeAnd Z
_FourAre each independently absent, or one or
Flanking DNA residues consisting of multiple nucleotides
And possibly connected,)
DNA encoding human GdNPF, DN of formula (IV)
Double-stranded DN consisting of A and its complementary DNA
A, the complementary DNA itself, one or more nucleologs
These mutants in which the tide is mutated, and 15 or more
With respect to these DNA fragments comprising
I do. The invention further provides the following formula (V): [0033] Embedded image[0034] Embedded image[0035] Embedded image(Where Z is_FiveAnd Z₆Are independent of each other,
Absent or one or more nucleotides
A flanking DNA residue consisting of
Is connected) to the rat GdNPF
DNA to be loaded, DNA of formula (V) and its complement
Stranded DNA comprising complementary DNA and complementary DNA
Itself has one or more mutated nucleotides.
Including these variants, as well as 15 or more nucleotides
These DNA fragments consist of: Furthermore, the present invention relates to a human Gd of the formula (I)
RNA encoding NPF or rat GdNPF; 1
Or more than one, in particular one, two, three or four nuclei
Those variants in which otide is mutated; and 15 or more
A fragment, in particular a species of said RNA comprising
Each Y has the above meaning, but the DNA residue is
Deoxythymidine (T)
Of the formula (III) in which is replaced by uridine (U)
R in formula (IV) wherein A and T are replaced by U
NA and RNA of formula (V). DNA encoding GdNPF and its mutation
Related polypeptides that maintain body and neurite-promoting activity
And the fragments of these DNAs are
E.g., cultivating the transformed host and
From the desired DNA or condensation of nucleotides
Can be prepared by chemical synthesis according to In particular, these DNAs are, for example, (a)
Poly (A) messenger RNA (mR
NA) and optionally encodes GdNPF.
Enriching the RNA or fragment thereof and reacting with the mRNA
To prepare a complementary single stranded DNA and from said single stranded DNA
Preparing double-stranded complementary DNA (ds cDNA);
Alternatively, (b) genomic DNA is isolated from appropriate cells and
To select the desired DNA using a DNA probe;
And (c) the ds cDNA of step (a), or
(B) Introduction of ds DNA into an appropriate expression vector
(D) suitable lodging depending on the obtained hybrid vector.
(E) GdNPF DNA or its fragment
GdNPF DNA or fragment thereof from a host containing no fragments
Selecting a transformed host containing the pieces; and
(F) isolating the desired DNA;
Can be. The polyadenylation messenger RNA is
It is isolated from known cells of the rearing line by known methods. This one
Methods include, for example, washing tissues with detergents and ribonuclease inhibitors,
For example, heparin, guanidine isothiocyanate and mer
Homogenize in the presence of captoethanol.
The dinate is centrifuged and the magnesium salt, e.g.
MRNA from supernatant with salt mixture containing gnesium
The precipitate is resuspended and the appropriate chloroform-
Depending on the phenol mixture, detergents and / or
Is extracted in the presence of a cationic chelator and
Salt-containing aqueous phase to ethanol, isopropanol, etc.
And to precipitate more mRNA. [0041] Alternatively, the mRNA may be cesium chloride.
Centrifugation in a gradient, followed by ethanol precipitation
Can be separated directly. Preferably,
The precipitated poly (A) mRNA is chromatographed,
For example, affinity chromatography, for example,
Go (dT) cellulose or oligo (dU) sepharose
Further purification by chromatography above. Glial system used for isolation of mRNA
Cells can be of various origins. For culture
Glioma cells that can be expanded
Glioma cells or rat nerves from isolated cell lines
Glioma cells are preferred. "Collection Nationale de Cultu
res de Microorganismes ", Institute of Pasteur, Paris,
No. Deposited on February 5, 1986 as I-518
Cells from the isolated human glioma cell line LN-340
Particularly preferred. In some cases, it is isolated from glial cells.
MRNA that encodes a selected neurite-promoting factor
You. This can be, for example, mRNA rate fractionation or chromatographic
Rough fractionation, with appropriate cells, such as frog oocytes or
Is a cell-free system, such as reticulocyte extract or wheat germ extract
Translation of said fraction in and neurite-promoting activity, or
Any other property that parallels the process promoting activity, such as professional
Of the resulting polypeptide for its inhibitory activity
It can be performed by cleaning. Polype obtained
Screening for peptides can be done using antibodies, such as polyclonal
Immunoassay using antibodies or monoclonal antibodies
A, for example, radioimmunoassay, enzyme immunoassay
Or in immunoassays using fluorescent markers
Especially effective. These immunoassays and poly
Preparation of clonal and monoclonal antibodies is an industry
Are well known in the
Can be used. Screening can also include hybridization
Performed at the mRNA stage using the
Additional steps for translation can be omitted. this
At least one such hybridization probe
Total synthetic DNA consisting of 7 nucleotides, or natural source
Or DNA isolated from genetically engineered microorganisms
Alternatively, it can be a DNA fragment. For example, Gd
Plasmid containing DNA encoding NPF
Amplify in a suitable host, then linearize and isolate DNA
You. Labels suitable for such DNA, such as radioactive labels
After adding the DNA, the DNA is used to generate the relevant GdNPF.
Is screened for mRNA encoding Preferably
Is the radiolabel that encodes rat GdNPF.
Hybridization probe consisting of isolated DNA
The presence of mRNA encoding human GdNPF
And screen the mRNA library. Preparation of the isolated DNA from the mRNA template
As with the preparation of double-stranded DNA from single-stranded DNA,
Well-known in mRNA template
A mixture of nucleoside triphosphates, in some cases
Radioactively labeled deoxynucleoside triphosphate
Plate (so that the results of the reaction can be screened),
Primer (e.g., poly (A) tail of mRNA)
An oligo-dT residue that hybridizes, and a suitable enzyme;
For example, incubate with reverse transcriptase. Mold mR
After denaturing the NA, the complementary DNA (cDNA) is
As described above, the mixture of deoxynucleoside triphosphate
And double-stranded D
Get NA. Suitable enzymes are reverse transcriptase, E. coli. Kori (E. FIG.
coliA) a Klenow fragment of DNA polymerase I,
Or T4 DNA polymerase. In some cases
Can be used to convert single-stranded DNA to the same deoxynucleotide
The same deoxynucleate complementary by extension by
It allows the use of nucleotide primers, but
Usually, the formation of ds DNA follows the natural hairpin formation.
Starts with This is the result of hairpin formation
Nucleic acid to convert hair DNA into SI nucleus
Further processing with ze. As an alternative to preparing cDNA from mRNA
Alternatively, the genomic DNA can be isolated and the desired polypeptide
Screening for DNA encoding the code
Can be. Genomic DNA is known in the art
According to the method, from appropriate tissues, for example, human placental cells or
Is isolated from human fetal hepatocytes. Follow established methods
With appropriate restriction enzymes such as AluI and HaeIII
Digest and lambda sharon phage, eg lambda sharon
Genomic DNA library
Prepare. Geno replicated on nitrocellulose membrane
A DNA probe, such as 17
A synthetic DNA probe consisting of more than two nucleotides, or
Is the cD from mRNA encoding the desired polypeptide
Screen using NA. Ds DNA prepared from mRNA or
Introduction of ds DNA derived from genome into an appropriate vector
Well known in the art. Preferably suitable
Cut the vector and use the same deoxynucleoside
A tail consisting of Ds to be annealed next
DNA has a complementary tail of the same deoxynucleoside.
Must be supported by the corresponding deoxy
Nucleoside triphosphates and enzymes, terminal
In the presence of oxynucleotidyl transferase
This is achieved by incubation. Otherwise
Ds DNA with the aid of a linker oligonucleotide.
And can be introduced by blunt end ligation.
You. The appropriate hybrid vector obtained from the hybrid vector
Transformation of a host is well known in the art. An example
For example, E. Coli in a medium containing calcium chloride.
Conditions for transformation by incubation
And then treated with the hybrid vector. Transformation
The identified host is then replaced with a suitable marker, such as an antibiotic resistance marker.
For example, tetracycline or ampicillin resistance
Select by. GdNPF DNA or a fragment thereof
The selection of transformed hosts with
It can be carried out. For example, the transformed host
Isolating the Smid DNA and immobilizing it according to standard methods;
Next, the first glyph used as a template for the cDNA was
Hybridize with total mRNA from A-lineage cells.
Elute the hybridizing RNA and proceed as described above.
And translate in vitro. The polypeptide obtained after the translation is neurite
Stimulatory activity, protease inhibitory activity, or anti-GdNP
Screening for an immune reaction with the F antibody, and
Selecting a transformed host containing the corresponding DNA
You. Alternatively, the total DNA of the transformed host is
Hybridize with DNA encoding dNPF
You. For example, it is predicted to encode human GdNPF.
The DNA of the transformed host is purified from rat GdNPF.
A DNA probe known to encode
Let it hybridize. The production of the DNA of the present invention can also be carried out by chemical synthesis.
Can also be done. For synthesizing DNA
A suitable method is S. A. Narang [Tetrahedron,39, 3
(1983)]. Known
Of synthetic polynucleases up to 40 nucleotides in length
Preparation of leotide requires good yield, high purity and relatively short
Make it possible in time. Properly protected nucleotides,
Phosphodiester method (K.L.Agarwal et al.,Angew. Che
m.,84, 489 (1972)] or a more efficient
The sophotriester method [C. B. Reese,Tetrahedron,3
4, 3143 (1972)], phosphite trieste
Method (R.L.Letsinger, etc.J. Am. Chem. Soc.,9
8, 3655 (1976)], or the phosphoramidite method
(S. L. Beaucage and M.H. Caruthers,Tetrahedron
Letters,22, 1859 (1981)]
Link. Oligonucleotides and polynucleotides
Can be simplified by the solid-phase method.
In, the nucleotide chain is attached to a suitable polymer
Confuse. Itakura etc.J. Am. Chem. Soc.,103, 70
6 (1981)] is a triad instead of individual nucleotides.
Nucleotides, and these are
They are linked in the solid phase synthesis by the tell method. Like this
Up to 67 nucleotides in a short time and with good yield
Can be prepared. The actual two
The strand DNA is chemically synthesized from both DNA strands.
Enzymatically assembled from overlapping oligonucleotides
You can stand up. In this case, the oligonucleotide is base-paired.
Is retained in the correct orientation by the
Are chemically linked together. Another possibility is two D
Overlapping single polynucleotide from NA chain
The sequence is composed of the four required deoxynucleoside triphos
In the presence of fate, a DNA polymerase such as DN
A polymerase I, Klenow cleavage of polymerase I
Pieces, or with T4 DNA polymerase, or A
In with MV (avian myeloblastosis virus) reverse transcriptase
Including cubating. This allows two polynus
The nucleotide sequence is maintained in the correct sequence by base pairing.
And complemented with the required nucleotides by the enzyme
To obtain a complete double-stranded DNA [S. A. Narang et al.
Anal. Biochem.,121, 356 (1982)]. The present invention further relates to GdNPFs and related peptides.
DNA encoding a peptide or a fragment thereof;
In some cases, including those linked to expression control sequences
And a method for producing the same.
You. The vector is the expected host cell for transformation.
Is selected depending on Examples of suitable hosts are restriction enzymes or
Is a microbe that lacks or has a low content of modifying enzymes
Object, such as yeast, such as Saccharomyces cerevisiae
(Saccharomyces cerevisia
e) And bacterial strains, especially Escherichia coli (Esc
herichia coli) Strains such as E. Stiff
(E. FIG. coli) $ 1776; Coli HB101,
E. FIG. Coli W3110, E.C. Coli HB101 / LM103
5, E. E. coli JA221 or E. coli. Coli K12 strain 294,
Bacillus subtilis (Bacillus subti
lis), Bacillus stearothermophilus (Bac
illus sweetarothemophilu
s), Pseudomonas (Pseudomonas), F
Mofilus (Haemophilus), Streptocco
Cass (Streptococcus) And more
Cells of higher organisms, especially established human or animal cells
Line. E. FIG. The above strains of E. coli, such as E. coli. Coli HB
101 and E.P. Kori JA221 and Saccharo
Mrs cerevisiae is preferred as the host microorganism. In principle, replicate in the chosen host,
And all of the GdNPF genes of the present invention are expressed.
Vectors are suitable. E. FIG. GdNPF in coli strains
Examples of suitable vectors for the expression of
A phage, such as a derivative of a λ bacteriophage, or
Plasmids, such as in particular plasmid ColEl and its
Derivatives such as pMB9, pSF2124, pBR31
7 or pBR322. Preferred of this invention
The vector is derived from the plasmid pBR322. Appropriate
Vector depends on complete replicon and expression plasmid
Selecting the transformed host based on the phenotypic trait and
Contains a marker gene that allows for identification.
Appropriate marker genes are available to the host, such as precious metals, antibiotics
Provides resistance to quality, etc. Further, a preferred vector of the present invention is
Restriction end besides replicon and marker gene region
Contains recognition sequences for nucleases, thereby
The GdNPF gene and, where appropriate, expression control sequences
It can be inserted into these sites. preferable
The vector, plasmid pBR322 and the induced
Plasmids, such as pUC9, are used for intact replicons,
Confers resistance to tracycline and ampicillin
Marker gene (tet)^RAnd amp^R)
Restriction endonucleases such as PstI (amp^RHeredity
Cleave the child, tet^RLeave the gene intact
BamHI, HindII, and SalI (these
Are all tet^RCleavage in the gene, amp^RHeredity
Leave the child intact), for NruI and EcoRI
Contains many unique recognition sites. Several expression systems for controlling gene expression
An array can be used. Especially transformed
Expression control sequences of highly expressed genes
Used. Using pBR322 as a hybrid vector
And E. coli as a host. When using stiffness, for example
For example, lactose operon, tryptophan operon,
Expression control sequences such as the rabinose operon (especially promoter
And ribosome binding sites), β-lactamers
Expression control sequence, phage λN gene and
Corresponding sequence such as gene fd-coat protein gene
It is. Plasmid pBR322 is a β-lactamase gene.
Contains promoter of gene (β-lac gene)
However, other expression control sequences must be introduced into this plasmid.
Must. Suitable for replication and expression in yeast
Vectors are yeast origins of replication and selectable genetic markers for yeast
Contains Origin of yeast replication, for example, chromosome autonomous replication
Hybrid vector containing segments (ars)
Is extrachromosomally retained in yeast cells after transformation.
Autonomous replication. Furthermore, it is homologous to the yeast 2μ plasmid.
It is possible to use a hybrid vector containing the sequence.
Wear. Such a hybrid vector is already in the cell.
Will be recombinantly integrated into the existing 2μ plasmid.
Would. The 2μ sequence is a plasmid of high transformation frequency.
Particularly suitable for and allows for high copy numbers
You. The preferred yeast vector of this invention is plasmid pJ.
DB 207. Suitable marker genes for yeast are notably
To confer antibiotic resistance to the host, or nutritional requirements
In the case of a yeast mutant, the gene complements host injury.
You. The corresponding gene is, for example, the antibiotic cyclohexyl.
Confer resistance to mids or to auxotrophic yeast mutants
It gives nutrition, for exampleURA3,LEU
2,HIS3Or especiallyTRP1Is a gene. Yeast yeast
The hybrid vector is further preferably a bacterial host, especially
E. FIG. Contains origin of replication and marker genes for E. coli
Construction and hybridization of hybrid vectors and their intermediates
To be performed in a bacterial host
I have. Expression Control Suitable for Expression in Yeast
The sequence is, for example, a highly expressed yeast gene. You
That is,TRP1gene,ADHIOrADHIIHeredity
Offspring, acid phosphatase (PHO3OrPHO5) Heredity
Offspring, each promoter of the isocytochrome gene, or
Is the promoter of the gene for the enzyme involved in glycolysis, for example
Enolase, glyceraldehyde-3-phosphate
Dehydrogenase (GAPDH), 3-phosphoglycele
Kinase (PGK), hexokinase, pyruvate
Decarboxylase, phosphofructokinase, gluco
Ose-6-phosphate isomerase, 3-phosphogly
Serate mutase, pyruvate kinase, triose
Sulfate isomerase, phosphoglucose isomerer
Uses promoters for zeo and glucokinase genes
can do. Preferred vectors of the present invention are
Promoters with transcription control, for examplePHO5,ADHII
as well asGAPDHAre promoters of genes, these are
Turn on or off due to changes in growth conditions
Can be. For example,PHO5The promoter is
Just increase or decrease the concentration of inorganic phosphate
Can be suppressed or unsuppressed. [0061] Suitable for replication and expression in mammalian cells.
The vector is preferably a virus, such as Simian
Virus 40 (SV40), Rous sarcoma virus (RS
V), adenovirus 2, bovine papilloma virus (BP)
V), papovavirus BK
Mutant (BKV) or mouse or human cytomega
It has DNA from the rovirus (CMV). Preferably
Such vectors are compatible with eukaryotic transcription control sequences.
E. Origin of replication and antibiotics for amplification in E. coli
Contains a quality resistance gene. In particular, the so-called shuttle vector
The pBR322E. Coli plasmid and SV40
And / or CMV enhancer and promoter regions
Can be created from For example, the plasmid may be a mouse or human
Itomegalovirus major early gene (major im)
enhancer of mediate-early gene)
-Unit, combined with human α-globin promoter
SV40 enhancer and / or further induced
Sexual promoters, such as heat shock genes or meta
Containing a promoter derived from the rothionein gene
Can be. In addition, it is usually related to the gene sequence of interest
Promoters or control sequences can be used. Duplicate
The production starting point is a heterologous replication starting point, such as SV40 or other
Vector containing an origin of replication from the viral origin
Or the chromosomal duplication of the host cell.
Provided by the manufacturing mechanism. Vector is transferred to host cell chromosome
When incorporated, the latter method is more effective. In a preferred embodiment, the invention relates to a host
Hybrid vector capable of replication and phenotype selection in strains
This vector contains a promoter and GdNP
F, DNA encoding related peptides or fragments thereof
This DNA is a transcription start and stop signal.
And the hybrid vector together with the translation initiation and termination signals.
Transformation under the control of the promoter
It is expressed in the selected host to produce the polypeptide.
It is arranged so that it is. The invention may be further transformed
This method relates to a method for producing a host, the method comprising:
Expression vector containing the DNA of the invention controlled by the
And transforming with a vector. This invention is
Furthermore, it relates to the transformed host itself. Examples of suitable hosts include the microorganisms described above, for example,
Calomyces cerevisiae, Bacillus subtilis,
And Escherishi Kori. Expression plus of the present invention
Transformation with mids, for example, as described in the literature
It is done. S. A. Hinne for cerevisiae
nc, Proc. Natl. Acad. Sci. USA,75, 1929
(1978). Anagnostopou For Subtilis
los, etc.J. Bacteriol.,81, 741 (1961)
And E. For M. Mandel et al.J. Mol.
Biol.,53, 159 (1970). That is, E. How to transform E. coli cells
Cell Ca to allow uptake of DNA⁺⁺place
And incubation with hybrid vectors
Including. Separating transformed cells from parent cells
Transfer cells to selective growth medium to allow. Contains vector
No cells will not survive in such a medium. Yeast
The transformation of the mother is performed, for example, by (1) glucosidase
Enzymatic removal of yeast cell wall, (2) polyethylene glycol
And Ca⁺⁺Sphering with vectors in the presence of ions
Treatment of loplast and (3) spheroplast agar
Regenerating the cell wall by embedding in the cell.
Preferably, the regeneration agar is used for regeneration of the transformed cells.
It is prepared to allow for simultaneous selection. Another example of a suitable host is a mammalian cell, such as a mammalian cell.
COS-7 cells, Bowes melanoma cells, Chiney
Zuhamster ovary (CHO) cells or fetal lung cells L-
132. Vectors are helper compounds, such as di
Ethylaminoethyl dextran, dimethyl sulfoxy
, Glycerin, polyethylene glycol, etc.
Or co-precipitate the vector DNA with calcium phosphate
And transfected into mammalian cells
Is done. Another suitable method is to use vector DNA into the cell nucleus.
Microinjection and electroporation (select
, ie increase the permeability of the cell membrane
It involves the introduction of DNA by a short pulse applied. Subsequent selection of the transformed cells is as follows:
Integrated covalently into the expression vector or separately
This can be done using a selection marker that has been added.
Selectable markers include antibiotics such as G-418 (Neoma
Confers resistance to isin) or hygromycin
Gene or host injury, such as thymidine kinase
Or hypoxanthine phosphoribosyltransferase
The gene that complements the absence of is included. Transformed
Host cells contain assimilable carbon and nitrogen sources and inorganic salts.
In a liquid medium having a method known in the art
Incubate. The culture of the transformed host of the present invention
Various carbon sources can be used for this. Preferred charcoal
Examples of sources include assimilable carbohydrates, such as glucose,
Maltose, mannitol or lactose, or vinegar
Acid salts, which can be used alone or in suitable mixtures.
Can be used. Amino acids as examples of suitable nitrogen sources
Acids such as casamino acids, peptides and proteins and the same
Degradation products such as tryptone, peptone or meat
Kiss, and also yeast extract, malt extract, and more
Ammonium salts such as ammonium chloride, ammonium sulfate
And ammonium nitrate, which are used alone
Or as a suitable mixture. Use
Inorganic salts that can be used include, for example, sodium and potassium.
Lium, magnesium and calcium sulfates, chlorides
Substances, phosphates and carbonates. The medium may further comprise, for example, a growth promoting substance, eg,
For example, trace elements such as iron, zinc, manganese, etc.
Alternatively, it exerts selection pressure and loses the expression plasmid.
Contains a substance that prevents the growth of cells. That is, an example
For example, if the expression plasmid is amp^RWhen containing a gene
, Add ampicillin to the medium. This is an antibiotic
Addition also removes antibiotic-sensitive contaminating microorganisms.
It has the effect of being done. For example, essential amino acid nutrition
When a yeast strain that is auxotrophic is used as a host microorganism,
The plasmid preferably contains an enzyme that complements the host defect.
Contains the gene to be loaded. The yeast strain is cultivated using the amino
Performed in minimal medium lacking acid. The vertebrate cells may contain a growth promoting substance and / or
Use commercial media that may be supplemented with mammalian serum
To grow under tissue culture conditions. Cells to a solid support,
For example microcarriers or porous glass fibers
Free floating in a suitable culture vessel.
Let it grow. Culture is performed by methods known in the art.
Do more. Culture conditions such as temperature, medium pH, and
The fermentation time will give the highest titer of the polypeptide of the invention.
To choose. That is, E.I. E. coli or yeast strains are preferred.
Preferably, aerobic culture is performed by submerged culture with shaking or stirring.
At a temperature of about 20 ° C. to 40 ° C., preferably about 30 ° C.
At a pH value of 4-8, preferably about 7.
About 4 to 30 hours, preferably the polypeptide of the present invention.
Cultivate until maximum yield is achieved. When the cell density reaches a sufficient value, the culture is stopped.
Stop and isolate the polypeptide. Polypeptide
When fused to the appropriate signal peptide sequence, this
Secreted directly into the supernatant by the cells. Otherwise
Is a detergent such as SDS, NP-40, Triton or
Crush cells by treating with oxycholic acid
Or lysozyme or a yeast having a similar effect
Cells must be lysed by elemental or ultrasound. Inn
When using yeast as the main microorganism, use glucosidase
The cell wall can be removed by the enzymatic digestion used
You. Alternatively or additionally, a mechanical force, such as
Shearing force (for example, X-press, French press,
) Or with glass beads or aluminum oxide
Shaking or, for example, freezing in liquid nitrogen and e.g.
Destruct cells using repeated thawing at 30 ° C to 40 ° C
Can be Centrifugation of the mixture obtained after disrupting the cells
Proteins, nucleic acids and other cellular components obtained after separation
The contained cell supernatant or solution can be prepared in a manner known per se.
A protein containing the polypeptide of the present invention.
Concentrate. That is, for example, most non-proteinaceous
Is removed by polyethyleneimine treatment and this
Proteins including the light polypeptides can be
Precipitation by saturation withium or other salts. other
In the above method, the cell supernatant or the lysate is chromatographed.
It can be directly pre-purified by the フ method. Additional purification steps include, for example, ultrafiltration,
Afiltration, gel electrophoresis, chromatography
Methods, such as ion exchange chromatography, size exclusion
Chromatography, high pressure liquid chromatography (H
PLC), reverse phase HPLC, immobilized polypeptide liquid chromatograph
To a suitable gel filtration column such as tomography (FPLC)
Separation, dialysis, and
Affinity chromatography, e.g., for antibodies, especially
Affinity chromatography using noclonal antibodies
Fees and other methods known in the art.
Include. The invention is further conditioned by the method of the invention.
GdNPF, related peptides and their fragments
Related. The invention particularly relates to the hybrid vehicles described in the examples.
Vector, transformed host cells, human GdNPF, and
And their manufacturing methods. The GdNPF of the present invention, related peptides and
Neurite-promoting and serine proteases of those fragments
Due to their inhibitory properties, these polypeptides can cause damage to the nervous system.
Useful for promoting nerve fiber regeneration later and preferred
Alternatively, a therapeutically effective amount of the active ingredient may be administered to an inorganic or organic individual or
Is a pharmaceutically acceptable drug suitable for liquid parenteral administration.
Used in the form of a pharmaceutical preparation comprising a carrier
You. Parenteral preparations are particularly useful in various ways, for example, intravenously, intramuscularly.
Applicable for intramuscular, intraperitoneal, intranasal, intradermal or subcutaneous administration
It is an effective injection. Such a solution is preferably active.
The active ingredient alone or with a pharmaceutically acceptable carrier
Prepared before use from lyophilized preparations
Isotonic aqueous solution or suspension. Pharmaceutical preparations are sterile
And / or auxiliaries, such as preservatives,
Stabilizer, wetting agent and / or emulsifier, stabilizer, osmotic pressure adjustment
Salts and / or buffers may be included. The pharmaceutical preparation of the present invention may optionally contain other drugs.
Contains substances of physical value and is known per se
Manufactured by conventional dissolution or freeze-drying methods
And about 0.1 to 100%, especially about 1% to about 50
% Active ingredient, and 1 in the case of a lyophilizate
Contains up to 00% active ingredient. This invention also provides
The pharmaceutically active compound of the invention is pharmaceutically acceptable
Manufacture of a pharmaceutical preparation characterized by being mixed with a carrier
About the method. The method of administration and dosage of the characteristics will depend on the patient
Selected by a physician taking into account the condition, type of disease and disease state
Will be selected. For example, nervous system injury occurs once a day
Or 0.001-1 mg / two times daily for several weeks
Treated by administration of kg body weight. Next, by way of example,
The following is a more specific description of the present invention.
It does not limit the scope. Abbreviations used in examples
Has the following meaning: [0076] bp base pairs BSA bovine serum albumin cDNA complementary DNA cpm count / min (radioactive decay) dA 2'-deoxyadenosine dATP 2'-deoxyadenosine triphosphate dC 2'-deoxycytidine dCTP 2'-deoxycytidine triphosphate dG 2'-deoxyguanosine dGTP 2'-deoxyguanosine triphosphate DMEM Dolbeco's Modified Eagle Medium DNA deoxyribonucleic acid dNTP Mixture of dATP, dCTP, dGTP and dTTP dpm decay / min (radioactive decay) ds DNA Double-stranded DNA [0077] dT 2'-deoxythymidine DTT 1,4-dithiothreitol dTTP 2'-deoxythymidine triphosphate EDTA ethylenediaminetetraacetic acid FCS fetal calf serum GdNPF glial-derived neurite-promoting factor Hepes N-2-hydroxyethylpiperazine-N'-2-ethanesul                 Honic acid mRNA messenger RNA PBS phosphate buffered physiological salt solution Pipes piperazine-N, N'-bis (2-ethanesulfonic acid) PMSF phenylmethylsulfonyl fluoride RNA ribonucleic acid rpm rpm / min SDS sodium dodecyl sulfate TFA trifluoroacetic acid Tris Tris (hydroxymethyl) aminomethane tRNA transfer RNA The following buffers and media are used. Elution buffer 10 mM Tris-HCl, pH 7.5, 1 mM EDTA,                   0.2% SDS. Immune buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA,                   150 mM, NaCl, 0.1% Tween-20^TM. Laemmli's 62.5 mM Tris-HCl, pH 6.8, 2% SDS, Sample buffer 10% glycerin, 5% 2-mercaptoethanol,                   0.001% bromophenol blue. LB-medium 1% Bacto tryptone (Difco), 0.5% Bacto yeast                   Extract (Difco) with 170 mM NaCl, NaOH                   Adjusted to pH 7.5. [0079] RVT buffer 200 mM Tris-HCl, pH 8.3 at 42 ° C,                   20 mM MgCl_Two, 280 mM KCl, 20 mM DTT. SSC buffer 15 mM sodium citrate, 150 mM NaCl, NaOH                   Adjusted to pH 7.0 with. TBE buffer 89 mM Tris (TRIZMA® base),                   89 mM boric acid 1 mM EDTA. TNE buffer 10 mM Tris-HCl, pH 8.0, 1 mM EDTA,                   0.1 M NaCl. Wash buffer 10 mM Tris-HCl, pH 7.5, 1 mM EDTA,                   0.5M NaCl, 0.2% SDS. [0080]Example 1 M from rat C6 glioma cells
RNA isolation Rat C6 glioma cells (ATCC No. CCL 10
7) was replaced with 15 ml of Dulbet supplemented with 10% fetal bovine serum.
10 cm tissue containing modified Eagle medium (Gibco)
Grow in culture dish. 120 confluent
Culture dish (about 2.5 × 10⁷Rat glia
Tumor cells). D. Palmiter [Bi
ochemistry,13, 3606-3615 (1
974)] to concentrate mRNA.
I do. MRNA fraction obtained by magnesium precipitation
Further, M. Edmonds etc. [Proc. Natl. A
cad. Sci. USA,68, 1336─1340
(1971)] and oligo (dT) cellulose.
On the plate. [0081]Example 2. MR for rat GdNPF
Concentration of NA 600 μg of poly (A) RNA from Example 1 (1 μg / μ
1) Heat at 70 ° C for 5 minutes and cool quickly in ice water
And 10 mM Hepes pH 7.5 / 1 mM EDT
A / 5-20% sucrose wire in 100 mM NaCl
Load onto a linear gradient, and switch SW27 at 20 ° C.
Centrifuge at 2000 rpm for 23 hours in a rotor. 20 strokes
Collect the minutes and precipitate with ethanol at -20 ° C overnight.
Let it settle. mRNA in SS-34 rotor for 150
Centrifuge at 00 rpm for 20 minutes and add 1% in 75% ethanol.
Wash once and finally dissolved in water to a concentration of 1 μg / μl
I do. The individual fractions were collected as follows:
Assay by injection into cells. Each tested
Inject 50 nl of the mRNA fraction into one oocyte. same
100 μl group consisting of three eggs containing the same mRNA
For 24 hours at 22 ° C in Barth's solution
To The incubation medium was removed and
Guenther et al. [EMBO J.C.,4, 1963-
1966 (1985)].
Protease inhibitory activity in degradation (this is GdNPF
Assay (parallel to activity). In summary,
Milk powder suspension, human urokinase, purified human
Mixture of toplasminogen and the solution to be tested
Is incubated in the appropriate buffer mixture and 4
Casei by measuring the decrease in turbidity at 05 nm
Monitor decomposition. The following result is obtained. [0083] [Table 2] Fractions 13 and 14 were pooled and subjected to cDNA synthesis.
(Example 3). [0084]Example 3 D encoding rat GdNPF
Preparation of s cDNA Concentrated mRN encoding rat GdNPF of Example 2
Using A as a template, a Maniatis Handbook [T. Man
iatis, E.F.Fritsch and J. Sambrook, “Molecular Cl
oning, A Laboratory Manual ”, Cold Spring
-Laboratory, 1982]
Double-stranded DNA (ds DNA) by substantially the same method
Is prepared. 3.1.First-strand synthesis 100 mM Tris-HCl (pH 8.2, at 42 ° C)
), 70 mM KCl, 10 mM MgCl_Two, 1 mM D
TT, 1 mM each of dGTP, dCTP and dTTP,
0.4 mM dATP, 50 μCi α-³²P-dATP
(Amersham, 3000 Ci / mmol), 50 μg / ml
Rigo dT (PL biochemicals), 100 μg / ml
  mRNA (Example 2) and 50 units of avian myeloblast
Disease virus (AMV) reverse transcriptase (Stalin, Base
, Switzerland) at 42 ° C.
And incubate for 60 minutes. This solution is 10 mM
Stop the reaction by adjusting to EDTA. This mix
Add 1M NaOH to a final concentration of 0.2M
Hydrolysis at 52 ° C. for 90 minutes. 1MT
Neutralized with ris-HCl (pH 8.0) and 1M HCl
And then extract the reaction mixture with phenol / chloroform
I do. The organic phase was washed with 10 mM Tris-HCl (pH
8.0) with 1 mM EDTA / 100 mM NaCl
Back-extract once. Sephadex G pooled water phase
Applies to -50 columns. Collect 100 μl fractions
And Serencos count [P. W. J. Rigby and others,J.
Mol. Biol.,113, 237-251 (197)
7) Monitor the radioactivity according to the above. cDNA-containing fraction
Are pooled and precipitated with ethanol. Single chain c
The yield of DNA is 4.6 μg. 30 mM NaOH /
Charge on 2% alkaline agarose gel in 1 mM EDTA
The size of the cDNA was 7 as determined by electrophoretic mobility.
It is between 00 and 2000 nucleotides in length. 3.2.Second strand synthesis and S1 digestion The resulting cDNA was transformed into a final volume of 100 μl of 100 mM
  Hepes (pH 6.9), 10 mM MgCl_Two, 2.
5 mM DTT, 70 mM KCl, 0.5 mM each dNT
P, 5.8μCi^ThreeH-dGTP and 32 units of D
NA polymerase (Klenow fragment, Boehringer
Incubate at 20 ° C for 150 minutes in Mannheim
I do. Add 16 units of enzyme and incubate
Continue at 37 ° C. for 60 minutes. 10 mM EDTA
To stop the reaction. This mixture
Is extracted with phenol / chloroform and ethanol
And let it settle. The resulting DNA was purified with 250 mM NaCl, 5
0 mM sodium acetate (pH 4.5), 1 mM ZnSO_Four,
And 500 units / ml of S1 endonuclease (be
-Lingermanheim)
Incubate for 30 minutes at 37 ° C in 100 μl of the mixture
To The reaction was performed by adding EDTA to 10 mM.
Stop. Add 1M Tris-HCl (pH 8.0) to 10
After addition to 0 mM, the reaction mixture was phenol / chlorophore.
Extract with LUM. cDNA was converted to -7 with ethanol.
Settle at 0 ° C. for 30 minutes. After centrifugation,
Ds cDNA was converted to 50 mM Tris-HCl (pH
7.4), 7 mM MgCl_Two, 1 mM DTT, 1 mM each
DNTPs and 1.5 units of Klenow fragment
Dissolve in 20 μl of the containing solution. The reaction mixture was incubated at 22 ° C. for 30 minutes.
Bate and then add EDTA to 10 mM
To stop the reaction. Extraction and 10 mM Tris-
HCl (pH 7.5) / 1 mM EDTA / 300 mM Na
After passage through a Sephadex CL-4B column in Cl,
Aliquots of cDNA-containing fractions on 2% agarose gel
Assay with. Pool fractions containing molecules of 600bp or more
And precipitated with ethanol and 10 mM Tri.
Dissolve in s-HCl (pH 7.5) / 1 mM EDTA
You. 0.6 μg of ds cDNA is obtained. [0090]Example 4. D encoding rat GdNPF
from plasmid pBR322, containing s cDNA
Preparation of the vector of Transformation of E. coli HB101 4.1.Ds cDNA with poly (dC) tail
Preparation of 540 ng of the ds cDNA of Example 3 was replaced with 200 mM cacodyl
Potassium salt (pH 6.9), 1 mM CoCl_Two, 260pm
ol^ThreeH-dCTP and 6 units of terminal deoki
Synnucleotidyltransferase (PL Biochem)
Mycalus) at 37 ° C. in 100 μl of a solution containing
Incubate for minutes. EDT to stop the reaction
Add A to 10 mM. Phenol / chloro
Extract with form and precipitate the DNA with ethanol.
Confuse. cDNA with dC-tail at 10 ng / μl
Store at -20 ° C. 4.2.Cutting with poly (dG) tail
D with truncated pBR322 and poly (dC) tail
Annealing with s cDNA and resulting vector
E. E. coli transformation 33 ng of dC-tailed cDNA from Example 4.1
And pBR322 with 170 ng dG-tail (Ps
cut by tI, Boehringer Mannheim) and
Was mixed with 200 μl of 10 mM Tris-HCl (pH
7.5) in 100 mM NaCl / 1 mM EDTA,
Incubate at 65 ° C for 5 minutes and 55 ° C for 60 minutes.
And then cooled to 22 ° C. in a water bath. 40μ
l of this annealing mixture is mixed with Maniatis'
For transformation as described in the Handbook
200 μl of competent E. coli Coli HB1
Add to 01 cells. The mixture is kept on ice for 30 minutes and
And heat to 42 ° C. for 2 minutes, then treat with 1 ml of LB-medium
And incubate at 37 ° C. for 30 minutes. This
Of the LB-medium (200 μl per plate)
8 cm containing 20 μg / ml tetracycline
Naturally spread on the plate. Plate at 37 ° C for 16 hours
Incubate. About 5000 tetracyclines
A resistant colony is obtained. [0093]Example 5 Hybridization selected
DNA encoding rat GdNPF by translation
Identification of contained clones 5.1.Hybridization Individual colonies from Example 4 were selected using tetracycline at 20 μg / ml.
Increase to saturation in 200 μl LB-medium containing phosphorus
Let them breed. 50 μl to obtain a pool of 48 colonies
l of each preculture containing 20 μg / ml tetracycline.
Pipette into 100 ml of LB-medium. Pooh
Let them grow overnight. Maniatis Handbook
DNA was isolated using the alkaline lysis method described in
Let go. Plasmid DNA linearized with EcoRI
And extracted, and 10 mM Tris-HCl (pH 7.
5) Dissolve to a concentration of 0.1 μg / μl in 1 mM EDTA
Understand. Untreated 100 μl of this solution in 20 μl aliquots
GeneScreen ™ filters (New In
Pipette onto Grand Nuclea (1 cm)
And dry between stages. This filter was washed with 0.5M NaOH /
3MM ™ page immersed in 1.5M NaCl
Place on par (Whatman) four times for 1 minute each. This way
Was converted to 2M Tris-HCl (pH 7.4) / 2 times concentration S
On 3MM paper soaked in SC buffer and last
3MM paper immersed only in double concentration SSC buffer
Repeat on the paper. Next, the filter was doubled in S concentration.
Gently agitate in SC buffer and place in a vacuum stove at 80
Bake at 2 ° C. for 2 hours. Before use, remove the filter
Place in boiling water for 1 minute. From rat C6 glioma cells
Total RNA (Example 1) was converted to 30% formamide / 20 mM Pi
pes (pH 7.5) / 500 mM NaCl / 2 mM ED
Dissolve at a concentration of 2-3 mg / ml in TA / 0.4% SDS
You. Using 130 μl of this solution per filter,
Hybridize at 42 ° C for 17 hours with gentle stirring
Let me do it. The filter was washed with SSC buffer / 0.1% SD.
10 times at 60 ° C. in S and 5 mM Tris-HC
1 (pH 7.5) / 1 mM EDTA at 60 ° C once
I do. The hybridized RNA is removed from the filter by 2
00 μl of 5 mM KCl / 10 μg / ml + RNA (base
Ringer Mannheim, extracted with phenol)
Boiling followed by rapid drying in dry ice-ethanol
Elution is achieved by two cycles of rapid freezing. Thawing
Later, the eluates are pooled and the RNA is washed with ethanol.
Precipitate at -20 ° C overnight. Centrifuge the precipitate,
Then, it is washed in 75% ethanol. From each pool
Dissolve the obtained poly (A) RNA in 4 μl of water, and
In vitro translation and immunoprecipitation with anti-GdNPF antibody
Use for 5.2.In rabbit reticulocyte lysate
Vitro translation of Rabbit reticulocyte lysate (Amersham, N.90)
To³⁵S-methionine (Amersham, 1000 Ci / mmo
l) and dilution on ice with poly (A) RNA of Example 5.1
And 70% cell lysate³⁵S-methionine 2μCi / μ
1 and RNA to a final concentration of 4 μg / ml. The mixture
Incubate at 30 ° C. for 1 hour. 1 μl aliquot
At 0 and 60 minutes, respectively, and remove GF
Spot on the / C filter (Whatman), and
Trichloroacetic acid from Maniatis Handbook
Used for precipitation. For gel electrophoresis, 2-4 μl of cell lysis
Dissolve the lysate mixture with 20 μl of Laemmli sample buffer
, Boil for 2 minutes and 10% acrylamide
Analyze on gel [U. K. Laemmli,Natu
re,227, 680 (1970)]. Gel 30 ~
Run for 3-4 hours at a constant current of 40 mA. Solid
After fixing, the gel was treated with a fluorograph solution [ENLIGHT
NING (TM), New England Nuclear]
For 30 minutes, dry and preflash
Expose to Kodak X-5 film overnight at room temperature. 5.3.Specific for rat GdNPF
Of a typical polyclonal antibody About 50 μg of purified rat GdNPF [J. Guenth
er, etc.EMBO J.C.,4, 1963-1966 (1
985)] in Complete Freund's Adjuvant (Sigma)
Immunizes rabbits by subcutaneous and intramuscular injection
Sensitize. About 50 μg in incomplete Freund's adjuvant
Booster injection of purified rat GdNPF at 4 week intervals
Do with. Serum was collected 10 days after each booster injection.
R. Hankes et al. [Aml. Biochem.,1
19142-147 (1982).
Test for anti-GdNPF antibody using. Second post
Positive sera are obtained after booster injection. Of positive serum
Concentrate the immunoglobulin; Hjelm et al. [F
EBS Letter,28, 73-76 (197
2)] Standard protein A-Sepharose CL-
Purification using 48 (Pharmacia) chromatography
I do. 5.4.Synthesized by in vitro translation
Of rat GdNPF Incubated In Vitro Cell Lysis Mix of Example 5.2
15 μl of the mixture was added to 10 μg of the anti-GdNPF antibody of Example 5.3.
And 1 mM PMSF in 150 μl of immunobuffer
Mix with. Mixture of siliconized eppendle
Incubate at 4 ° C for 17 hours in a tube.
Protein A-Sepharose CL-4B (Pharmasi
A) by swelling the powder in PBS for 2-3 hours
Contains 2mg / ml BSA (Pentax, fraction V)
Washes three times in different immune buffers and containing BSA
Not freshly prepared by washing twice in the same buffer
I do. The Sepharose was diluted 1: 2 with the immune buffer.
And 15 μl of the cell lysate mixture and polyclonal solution.
In addition to the Eppendorf tube containing the
The suspension is then gently stirred at room temperature for 60 minutes.
You. This sepharose was added to 0.5% to 1%
Wash three times with Immune Buffer containing Peel-20.
After the final wash, 20 μl of Laemmli sample buffer
Pipette the solution into dry Sepharose pellets. Hanging
Incubate the suspension for 10 minutes at room temperature and finally
Boil for 4 minutes. Chilled on ice and centrifuged
Thereafter, 20 μl of the supernatant was loaded on a 10% polyacrylamide gel.
Load. Gel electrophoresis was performed and described in Example 5.2 above.
The color is developed by the method described above. Pool of 48 colonies
One of (Example 5.1) is positive. 4 of this one pool
Combine 8 colonies with 6 new pools of 8 colonies,
Then, the same processing is performed. Finally, positive immunity after translation
A single colony that gives a precipitate is identified. This clone
LB-medium containing 20 μg / ml tetracycline
Expand inside. 5.5.Rat GdNPF cDNA
Sequencing The cDNA of the positive clone of Example 5.4 was
Sequence using general methods. 9 μl of pBR3 in water
1.5 μg of the recombinant plasmid from Example 22 (Example 4.2)
PstI endonucleus of pBR322-derived vector
Immediately before the cleavage site, 1 μl of the complementary primer (0.
5 pmol). Heat DNA in boiling water for 3 minutes
Denatured in dry ice / ethanol
For 1 minute, thaw at room temperature, and add 1 μl of 10
0 mM Tris-HCl (pH 8.3) / 50 mM MgC
l_TwoMix with. This annealing mixture is heated at 37 ° C.
Incubate for 30 minutes. Amersham sequencing
Manual (M13 cloning and seq
uenching handbook, Amersham)
Perform the sequencing reaction as described in. Found
The resulting sequence is shown in formula (V). [0102]Example 6. Cloning of human GdNPF cDNA
Learning 6.1.Human glioma cells Dr. E.H.Macintype and N. de Tribolet (J.P.Perki
ns, etc.Life Sciences,Ten, 1069-1080 (1971); B. De M
uralt etc.Eur. J. Cancer Clin. Oncol.,twenty one, 207-216
 (1985)] established five human glioma cellular
The positive rat Gd of Example 5 was analyzed by Nasan blot.
In vitro-nick transcription of NPF cDNA clone
By slation³²DNA probe labeled with P
Analysis for the presence of mRNA that hybridizes with
You. Use 10 μg of total cytoplasmic RNA per lane.
Hybridization was performed at 42 ° C. for 15 hours, 1 × 1
0⁶Use cpm / ml nick translation probe
And do it. The filter was washed with 30 mM sodium citrate.
(PH 7.0) in 300 mM NaCl / 0.1% SDS
4 times for 5 minutes each at room temperature and 1.5 mM sodium citrate
Lithium (pH 7.0) / 15 mM NaCl / 0.1% SD
Wash twice in S at 60 ° C. for 15 minutes each. “Coll
ection Nationale de Cultures de Microorganisme
s ", Institute of Pasteur, Paris, 5 February 1986
Cell Line LN deposited with No. I-518
-340 gives a strong hybridization age. this
To confluence in 40 10 cm tissue culture dishes
Let them breed. 6.2.mRNA isolation About 10⁸Glioma cells containing three cells LN-3
1 ml of the 40 pellets is mixed with 100 g of guanidine thiocyanate.
Gin, 100 ml of water, 10.6 ml of 1 M Tris-H
Cl (pH 7.5), 4.2 ml of 0.5 M EDTA, 2
1.2 ml of 20% N-lauryl sarcosine and 2.1 ml
Filtered prepared from 2-mercaptoethanol
Dissolve in 6 ml of solution. 2.7g after shaking vigorously
Dry CsCl is added and the solution is added to a 12 ml centrifuge.
5.7 in 0.1 M EDTA (pH 7.5) in heart tube
Overlay on 2 ml of cushion solution consisting of M CsCl
You. Fill the tube with water and TST41 low
At 29000 rpm at 20 ° C.
Centrifuge for 16 hours. At the end of this run, most of the supernatant was removed.
And drain the tube quickly. Glassy R
The NA pellet was mixed with 0.4 ml of 10 mM Tris-HCl.
(PH 7.5) and sliding in 0.2% SDS and occasional addition
Dissolve by warming (2 minutes, 37 ° C). 1 ml of ethanol
And centrifuge in Eppendorf for 5 minutes
To precipitate the RNA. RNA (1.1
mg) in air and dissolved in 0.5 ml elution buffer
I do. Heated at 68 ° C. for 2 minutes and cooled on ice
Later, 55 μl of 5 M NaCl was added and the solution
Oligo-dT cellulose equilibrated in wash buffer
(Type 7, PL biochemicals) in a 2 ml column
Apply. After applying the sample three times in succession, the column
Was washed with 15 ml of wash buffer and the bound RNA
Is eluted with 4 ml of elution buffer. Eluted material
Heat at 68 ° C. for 2 minutes, cool, and add 0.44 ml
Add 5M NaCl. This solution is re-equilibrated
Apply to Rigo-dT cellulose column (3X). Fifteen
After washing with washing buffer of 4 ml, bound RNA was
Elute with elution buffer. 0.25 ml of 3M sodium acetate
By addition of lium (pH 5.5) and 10 ml of ethanol.
Precipitate the RNA overnight at -20 ° C. Precipitation (76
μg) by centrifugation (16000 × g for 15 minutes)
Collected, dissolved in 0.4 ml of water, and 25 μl of 3M
Reprecipitation by addition of sodium acetate and 1 ml of ethanol
Let it settle. After cooling in dry ice for 10 minutes,
RNA by centrifugation in Appendorf for 5 minutes
Collect Air-dry the pellet and put in 75 μl water
Dissolve. 6.3.Preparation of cDNA 10 μl (1 mg / ml) of the RNA solution was added to 25 μl RV
T buffer, 2.5 μl dNTP mixture (20 mM each)
ATP, dCTP, dTTP and dGTP), 5 μl
1 mg / ml oligo-dT_12-18(PL biochemical
S) 1 μl of α-³²P-dCTP (10 μCi, 300
0 / mmol), 3 μl of RNasin ™ (60 UNI
Biotech), 3 μl reverse transcriptase (66 units)
In a solution containing 2 μl of water) and 2 μl of water.
Incubate. The mixture is left at 42 ° C. for 1.5 hours
Incubate and then 2 μl of 0.5 M ED
Stop the reaction by adding TA (pH 7.5)
You. Add 25 μl of 0.15 M NaOH
And by incubating at 65 ° C for 1 hour
Degrades RNA. This solution was added to 25 μl of 1M Tr
is-HCl (pH 8.0) and 6 μl of 1 M HCl
Neutralize by adding. 2 μl of 20% SDS
And add this solution to 0.15 ml phenol / clo
Roform mixture (equal volumes of phenol and chloroform,
0.1% δ-hydroxyquinoline; 10 mM Tris-
HCl (pH 8.0), 1 mM EDTA and 0.1 M N
equilibrated with aCl]. The aqueous phase is buffered in TNE buffer in a Pasteur pipette.
Sephadex G-50 column 2 equilibrated with liquid
Apply to ml. Passage fraction containing 3 μg of cDNA
(0.4 ml) and add 1 ml of ethanol
And cool in dry ice for 10 minutes.
Precipitate NA. Centrifugation (Eppendorf, 5 minutes
After), dissolve the air-dried pellet in 32 μl of water
I do. The cDNA was replaced with 32 μl of cDNA (2.8 μl
g), 10 μl of 1 M potassium cacodylate (pH 7.
0) 5 μl of 10 mM CoCl_Two5 μl of 1 mM D
TT and 10μCi^ThreeH-dCTP (20 Ci / mmol, 1
Oligo-d in a reaction mixture containing
Extend by C-tail. Preincubation for 5 minutes at 37 ° C.
Followed by 3 μl of terminal deoxynucleotidyl tiger
Transferase (81 units, P-L biochemical
) And incubate for 10 minutes. 5
Add 0 μl of TNE buffer and add 0.1 ml of the solution
With a phenol / chloroform mixture. 0.1ml
Addition of ethanol and cooling in dry ice
Precipitate the cDNA. After centrifugation, pellet
Wash with 0% ethanol, air dry and 13 μl
Dissolve in water. 6.4.Preparation of sd cDNA 13 μl water, 25 μl RVT buffer, 2.5 μl
dNTP mixture (20 mM dATD, dCTP, d
TTD and dGTP), 5 μl of 0.2 mg / ml oligo-
dG_12-18(PL biochemicals), 3 μl α-
³²P-dCTP (10 mCi / ml, 3000 Ci / mmol),
And 3 μl of reverse transcriptase (66 units,
G) The above solution of the cDNA in
Incubate. 2 μl of 0.5 M EDTA (pH
7.5) and reaction by addition of 50 μl TNE buffer
And stop the mixture with 0.15 ml of phenol.
And a chloroform / chloroform mixture. The aqueous phase was separated with a Sephadex G-50 column (T
2.5 ml in NE buffer) and 1.8 μg
Collect the flow through containing the ds cDNA. 1 ml of d
Add ethanol and cool in dry ice.
This causes the DNA to precipitate. 32 resulting pellets
Dissolve in μl of water and for single stranded cDNA Example 6.
The DNA was oligo-ds
To extend. 1 μl of 0.5 M EDTA (pH
The reaction is stopped by adding 7.5) and
1% horizontal in TBE buffer using a 0.5 cm wide slot
Load sample on agarose gel. At 5V / cm
After 1 hour of electrophoresis, 1.5-2 Kb of appropriate size
The region containing the cDNA was excised and
-Put in a Collodion bag (Sartorius) and add water
Pre-soak inside. Add 0.3 ml of water and add the bag
Is an electrophoresis apparatus containing a 1/2 concentration of TBE buffer.
Put inside. Electrify DNA at 5V / cm electrode distance for 20 minutes
Electrophoresed, and recovered from the bag by vigorous pipetting.
Take it. 0.6 ml phenol / chloroform mixture
After extraction, 40 μl of 3M sodium acetate (pH 5.
5) and 1.2 ml of ethanol are added and the solution is
Cool for 10 minutes in dry ice. Centrifugation (eppe
90 ng of the ds cDNA after
And collect 20 μl of 10 mM Tris-HCl
(PH 8.0) and dissolved in 1 mM EDTA. 6.5.Cutting with poly (dG) tail
Ds with truncated pUC9 and poly (dC) tail
Annealing with cDNA and the resulting plasmid
According to E. E. coli transformation 20 μl of cDNA (size fractionated material of Example 6.4)
90 ng) to 8 μl (100 ng) of oligo-dC_10-18Te
PUC9 DNA with Pharma (Pharmacia) and 1
Mix with 72 μl of TNE buffer and at 65 ° C. for 1
0 min, 1 hour at 46 ° C, 1 hour at 37 ° C and room temperature
For 1 hour. Annealed
Using the prepared cDNA-plasmid DNA,
traits as described in the atis handbook
Competent E. coli prepared for conversion Coli HB10
One cell (LM1035 strain) is transformed. 1 μl of annealed DNA was added to 200 μl
1 competent cell and place on ice for 30 minutes
Good. Perform this method 60 times. 90 second heat shock
And after 2 minutes ice cooling, 0.8 ml SOC per tube
Add medium and incubate the tube at 37 ° C for 60 minutes.
Lab. SOC medium is 2% bactotryptone,
0.5% yeast extract (all manufactured by Gibco), 10 mM N
aCl, 2.5 mM KCl, 10 mM MgCl_Two, 5mM
  MgSO_FourAnd 20 mM glucose. ink
After the inspection, put all tubes together and
Containing 50 μg / ml ampicillin. Three MacConkey agar plates (15 cm)
Plate on top. Plates at 37 ° C overnight
Incubate. 2500 pieces generated per plate
Pick up the recombinant on a nylon membrane and add 2 replicates
Make mosquitoes. Master plate at 4 ° C on agar plate
And store the replica Maniatis handbag
Colony hybridize as described in the
Process for zation. [0117]Example 7 Glioma cell-derived human cDNA
Of cDNA encoding rat GdNPF
Redidation Rat GdNPF obtained from the positive clone of Example 5 was
The cDNA insert from 1 μg of the plasmid to be
digestion with stI restriction endonuclease, agarose
Remove by gel electrophoresis and gel elution. Pure cd
NA insert (100ng) from Amersham
Use a translation kit (N.5000)
Make radioactive as directed by the supplier. This radioactive cD
NA probe is 5 × 10⁸Has specific activity of dpm / μg
You. The replica filter (example 6.5) is set to 0.
9M NaCl, 0.18M Tris-HCl (pH
8.0), 6 mM EDTA, 0.02% Ficoll 40
0.02% polyvinylpyrrolidone, 0.02% BS
A, 0.2% SDS and 50 μg / ml modified bovine thymus DN
Prehybridization in 100 ml of solution containing A for 2 hours
Let me do it. Hybridization is performed in sealed
Nick translated in a plastic bag
1 ml of the same solution containing the heat-denatured cDNA probe
Perform overnight in. After the hybridization, the filter
With 0.9 M NaCl, 0.18 M Tris-HCl
(PH 8.0), containing 6 mM EDTA and 0.2% SDS
Wash twice at 65 ° C. in 200 ml of solution, then
45M NaCl, 0.09M Tris-HCl (pH
8.0), containing 3 mM EDTA and 0.2% SDS
Twice with 200 ml of solution, and 0.15 M NaCl,
0.03 M Tris-HCl (pH 8.0), 1 mM E
In a 200 ml solution containing DTA and 0.2% SDS
Wash twice. Expose filter to X-ray film overnight
I do. Positives appear on both replica filters. Eleven positive colonies were grown and
To isolate these plasmid DNAs for restriction analysis.
You. About 1.5 × 10^ThreeContains nucleotide inserts
Two recombinant plasmids were selected and Maniatis
Analysis of restrictions described in the Handbook of
ger (M13 cloning and sequence
Ninging handbook, Amersham)
Is used to determine the entire coding sequence. Restriction analysis and sequencing
The outline of the analysis is shown in FIG. List all sequences in formula (IV)
You. One clone has W equal to A (hence X_TwoIs Ar
g) resulting in a DNA of formula (IV)
The loan is for W is ACAG (hence X _TwoIs Thr-G
ly) of formula (IV). [0121]Example 8 Plastic encoding human GdNPF
Synthesis of human GdNPF by E. coli containing sumid Of Example 7, which is shown to contain a DNA sequence of formula (IV)
One of the two clones was replaced with about 1 in tryptone medium.
Optical density (OD₆₅₀). Get cells and then
30 mM NaCl and 50 mM Tris-HCl (pH
Resuspend in 0.5 ml of an aqueous solution containing 8.0).
Lysozyme (Sigma) is added to 1 mg / ml. After 30 minutes at 0 ° C., the suspension was
Frozen in and thawed at 37 ° C., repeated 7 times
And then at 4 ° C. in an SS34 Solval rotor for 2 hours.
Centrifuge at 000 rpm for 20 minutes. Casey of Example 2
Degradation assay and the neurite outgrowth of Example 9 (o
(outgrowth) assay.
Human GdNPF was replaced with Guen for rat GdNPF.
ther etc.EMBOJ. 4, 1963-1966
(1985)].
Chromatography on Phosphorus-Sepharose CL-6B
And subsequent chromatographic chromatography on Affigel-Blue
Purify by luffy. [0123]Example 9 Neurite outgrowth assay 40,000 EDTA-removed in culture dishes (35 mm)
Mouse neuroblastoma cell clone NB_TwoA (ATC
C cell deposit number CCL 131) and 10
10% CO in DMEM containing% FCS_TwoContains
Incubate at 37 ° C in a humidified atmosphere. 16
After １８18 hours, the medium was incubated against 0.9% NaCl solution.
Thoroughly dialyzed, then sterilized with a filter and frozen
Contains 0.5% FCS (not heat-inactivated)
With DMEM. 22 hours later, final volume
Add 100 μl of the cell suspension of Example 8 in 2.0 ml of DMEM
I do. 2.5% glutaraldehyde in PBS after 4 hours
Stop assay by fixing cells by
You. The degree of morphological differentiation was determined by Monard et al. [Pr
oc. Nat. Acad. Sci. USA,70, 18
94-1897 (1973)].
And determined by a phase contrast microscope. [0124]Example 10. Novel human GdNPF and known
Relationship between rat GdNPF About 250 μg of rat GdNPF [J. Guenthe
r, etc.EMBO J. ,4, 1963-1966 (19)
85)], T. Ruegg etc. [Methods
in Enzymology,47, 111-116
(1977)] and carboxymethyl
Become The reaction mixture was dialyzed against 0.1% TFA,
Concentrate to 1ml by Servant Speed Bag Concentrator
And desalting by reversed-phase high-pressure liquid chromatography.
I do. The sample was placed in a wide hole C₈-RP column 0.4x
25 cm (Baccarboned RP 7105-0, J.T.
Baker) and 40% to 50% aqueous
With 0.1% TFA in a gradient of acetonitrile
Treat for 30 minutes. Acetonitrile from protein-containing fractions
Remove the torils and concentrate to 0.5 ml (Servan
Speed back concentrator). The resulting solution, 120
μl of 1M NH_FourHCO_Three, And 10 μl of 10^-3M
  3 μg of trypsin (Worthington) in HCl
Incubate at 7 ° C for 24 hours. The reaction mixture is
Acidify to pH 2 with FA and on the same column as above
By high pressure liquid chromatography on a column. A sample was prepared with 0.1% aqueous TFA, and
0.1% TFA in 0% to 50% aqueous acetonitrile
Treat with a gradient for 90 minutes. Chromatography
Different polypeps clearly separated in the first half of
Collect each fraction containing tide and concentrate to 20 μl
And then a commercially available gas-liquid sequencer (Applied
(Io Systems). The following sequence is found
It is. 1) Thr-Phe-Val-Ala-G
ly-Asp-Gly-Lys (sequence of human GdNPF)
In connection with 187-194, Ala^FiveReplace with Gly
ing); 2) Phe-Gln-Pro-Glu-Asn-Thr
-Lys (identical to sequences 178 to 184 of human GdNPF)
); 3) Thr-Ile-Asn-Ser-Trp or Th
r-Met-Asn-Thr-Met-Val-Pro
-Lys (related to sequences 257-268 of human GdNPF)
Do); 4) Ala-Ile-Val-Ser-Lys (human G
dNPF sequences 79-83); and 5) Ala-Asn @ Phe-Ala-Lys (human G
dNPF sequences 303 to 307). These results indicate that the isolated human cDNA is indeed Gd
Corresponds to the NPF coding sequence and should not represent artifacts
Is shown. [0128]Example 11 FIG. Synthesis of GdNPF fragment Merrifield (A. Marglin and R.B.Merrifield,Ann. R
ev. Biochem.,39, 841-866 (1970)]
Using the solid-phase method, with two minor modifications
Synthesize the peptide. That is, tert-butoxycal
Amino acids protected by bonyl are converted to dicyclohexylcarbo.
With the addition of diimide and hydroxybenzotriazole
Activated [W. Koenig and Geiger,Ch
em. Ber.103, 788-798 (197).
9)], and the resin is mixed with a swelling solvent and a shrinking solvent.
Wash alternately (L. Corley, D.M.Sachs and C.B.
insen,Biochem. Biophys. Res. Commun.,47, 1353-1
359 (1972)]. 11.1.Peptide synthesis method B. F. Gisin [Helv. Chem. Act
a.,56, 1476-1482 (1973)].
Chloromethylated polystyrene according to
Ren resin (Merrifield polymer, Fluka, Butch
, Switzerland). M. A. Juiller
reactions designed by at (Nestek SA, Switzerland)
Perform peptide synthesis manually in the vessel. Mixing is performed in the reactor.
This is done by blowing the element. The reaction cycle consists of the following steps. (A) 50% TFA / CH_TwoCl_Two5 minutes; (B) 50% TFA / CH_TwoCl_Two, 25 minutes; (C) CH_TwoCl_Two3 times for 2 minutes; (D) CHCl_Three3 times for 2 minutes; (E) Triethylamine / CHCl_Three1: 9, 5 minutes 2
Times; (F) CHCl_Three3 times for 2 minutes; (G) dimethylformamide (DMF), 3 times for 2 minutes; (H) Thymolar amount of tert-butoxycarboni in DMF
Protected amino acids and thymolar amounts of 1-hydroxy
Benzotriazole, after 1 minute, CH_TwoCl_TwoMedium 4 times
Dicyclohexylcarbodiimide for 60 minutes; (I) DMF, twice for 2 minutes; (J) CH_TwoCl_TwoTwice for 2 minutes; (K) CH_ThreeOH, twice for 5 minutes; (L) CH_TwoCl_Two3 times for 2 minutes; (M) a second with a 2.5 molar amount of the same protected amino acid
The second coupling, steps (d) to (e); (N) CH_TwoCl_TwoMedium 1% N-acetylamidazole and
And 10% acetic anhydride for 30 minutes. The following Nα-tert-butoxycarboni
(N-BOC) -L-amino acid [Bach
em) Fine Chemicals, Bubendors, Sui
Use N-BOC-L-alanine Nα-BOC-Nωnitro L-arginine N-BOC-L-aspartic acid-β-cyclohexyl
ester N-BOC-L-glutamine N-BOC-L-glutamic acid-γ-benzyl ester N-BOC-L-glycine Nα-BOC-N^im-Tosyl-L-histidine N-BOC-L-isoleucine Nα-BOC-Nε-2-chlorobenzyloxycarbo
Nyl-L-lysine N-BOC-L-phenylalanine N-BOC-L-proline N-BOC-O-benzyl-L-serine N-BOC-O-benzyl-L-threonine N-BOC-L-tryptophan N-BOC-L-valine. Protected with tert-butoxycarbonyl
Amino acids aparagine, glutamine, isoleucine,
Steps (h) and (m) for lorin and threonine
A large excess of 6 to 8 mol in the coupling of
You. During the cycle, after the addition of tryptophan residues,
2% 1,2-ethanedithio in protection steps (a) and (b)
Of the indole ring by adding
Work around. Dehydration of the carbodiimide function of glutamine
Before addition to step (h).
Pre-form riazole ester (0 ° C., 15 minutes
Interim) to minimize it. 11.2.Gln-Lys-Ala-L
ys-Ile-Glu-Val-Ser-Glu-As
p-Gly-Thr-Lys-Ala:3.5 in the reactor
5 g (1.92 mmol) of an alanyl-substituted resin (0.54 mm
ol / g resin) and using the corresponding amino acids
13 cycles are performed. Small aliquot of ninhydrin
Monitor peptide synthesis by analysis. Final peptide
-Resin in 35 ml of hydrogen fluoride / anisole (10: 1 V /
V) for 1 hour at 0 ° C. Hydrogen fluoride under reduced pressure
Remove at 0 ° C. Wash the resin three times with ethyl acetate
And then extract three times with 50 ml portions of 50% acetic acid.
The extract is lyophilized and the residue is used for Whatman preparative
Use 25 ml of Column Partisil 10 ODS-3
0.1% TFA was obtained by high pressure liquid chromatography.
(10 minutes), then use 0-50% acetonitrile
Purification using 0.1% TFA in gradient (60 min)
I do. Purify the pure product with a commercial gas-liquid phase sequencer (A
Amino acid sequencing on Pride Biosystems)
Characterize more. 11.3.His-Lys-Ala-L
ys-Ile-Glu-Val-Ser-Glu-As
p-Gly-Thr-Lys-Ala:Example of this protein
Prepare exactly as in 11.2. However, the last size
Converts glutamine to histidine in the cle. 11.4.Arg-Ser-Ser-Pro-Pro
-Trp-Phe-Ile-Val-Asp-Ser-
Phe:3.0 g (1.29 mmol) of phenyl in the reactor
The alanyl-substituted resin (0.43 mmol / g resin) was prepared.
And 11 cycles with the corresponding amino acids
Do. The cleavage, deprotection and purification of the product are described in Example 11.2.
Perform the method described above. However, an additional 2% 1,2-eta
Hydrogen fluoride / anisole mixture containing dithiol
use. 11.5.Lys-Ser-Arg-Phe-Gln
-Pro-Glu-Asn-Thr-Lys-Lys-
Arg-Thr-Phe:This protein was identical to Example 11.4.
It is prepared by the same method. 13 using the corresponding amino acid
Perform a cycle. [0135]Example 12. Pharmaceutical preparation for parenteral administration 200 μg of human GdNPF was added to 3 ml of 5N human serum
Dissolve in Bumin. Filter the resulting solution on a bacteriological filter
And filter the filtered solution under sterile conditions for 10 bars.
Divide into ials. Place vials preferably in a cool place, for example
Store at -20 ° C.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a double-stranded cD encoding human GdNPF.
1 shows an outline of restriction analysis and sequence analysis of NA.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 1/21 C12R 1:19) (72) Inventor Sergio Grol Switzerland, 4052 Basel, Chefchaouen Bruggerstrasse 26 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 15/09 A61K 38/00 C07K 14/48 C12N 1/21 BIOSIS (DIALOG)

Claims (1)

(57) [Claims] The following formula (I): Wherein Cys is optionally present in the form of a disulfide; X ₁ is hydrogen, an acyl group, of the formula (II): Met (-19)
-Asn-Trp-His-Leu (-15) -Pro-Leu-Phe-Leu-Leu (-10) -Ala
-Ser-Val-Thr-Leu (-5) -Pro-Ser-Ile-Cys (-1)-
A peptide residue represented by the formula (II) wherein 1 to 4 amino acids may be replaced by another amino acid, or a formula (II) comprising 1 to 18 amino acids from the carboxy terminus: ) Are optionally acylated; and X ₂ is Arg or Thr-Gly.] And optionally glycosylated. Human glial-derived neurite-promoting factor or its related peptide. 2. Cys is optionally present in the form of a disulfide and X _{1 comprises} hydrogen, an acetyl group, a peptide residue of formula (II) or 1 to 18 amino acids from the carboxy terminus of formula (II) The human glial-derived neurite-promoting factor or its related peptide according to claim 1, which is a fragment of a residue. 3. Cys is present in the form of a disulfide as the case, and X ₁ is hydrogen, a peptide residue of the Formula (II), a fragment comprising amino acids -18 of ~-1 position of the Formula (II), Pro-Ser -Ile-Cys-, Ser-Ile-Cys-, Ile-Cys-, or
The human glia-derived neurite-promoting factor of formula (I) or a related peptide thereof according to claim 1 or 2, which is Cys-. 4. 4. The human glial-derived neurite-promoting factor or a related peptide thereof according to claim 1, wherein X ₂ is Arg. 5. _{2. The} method according to claim 1, wherein X ₂ is Thr-Gly.
Item 4. The human glial-derived neurite-promoting factor or the related peptide thereof according to item 2, 2 or 3.